P53, MDM-2, BAX and BCL-2 and drug resistance in chronic lymphocytic leukemia.

Most antitumor agents exert their cytotoxic effect through the induction of apoptosis, and this process may be mediated through an elevation in p53 protein, with a subsequent increase in bax and decrease in bcl-2. p53 also increases mdm-2 expression and mdm-2 may then bind and inactivate p53. Cells from 31 patients with chronic lymphocytic leukemia (CLL) were treated in vitro with 2-chlorodeoxyadenosine (CdA), arabinosyl-2-fluoroadenine (F-ara-A), or chlorambucil (CLB) and drug sensitivity measured using the MTT assay. The protein levels of bax and bcl-2 were measured in CLL cells from 25 patients, and were found to be higher in leukemic cells than in normal B cells. The bcl-2 levels varied three-fold, the bax levels fifteen-fold, and the bax:bcl-2 ratios ranged from 0.44 to 2.91. The expression of mdm-2 mRNA was measured in CLL cells from 28 patients and was found to vary twenty-fold. However, no correlation was observed between drug sensitivity to CdA, F-ara-A, or CLB and the cellular levels of mdm-2 mRNA, or the protein levels of bax or bcl-2, or the bax:bcl-2 ratio. Treatment of CLL cells having wild type p53 with CdA, F-ara-A or CLB produced an increase in p53 protein and mdm-2 mRNA. This was not observed in cells having a p53 mutation, and these cells were highly resistant to both CLB and the nucleoside analogs. In contrast to the nucleoside analogs and CLB, dexamethasone and vincristine had no effect on mdm-2 mRNA levels. Treatment of CLL cells containing a wild type p53 gene with CdA, F-ara-A, or CLB, did not produce any consistent changes in bax or bcl-2. Thus, CdA, F-ara-A and CLB appear to act in CLL cells through a p53-dependent pathway, whereas this does not occur with dexamethasone or vincristine. The cellular levels of mdm-2, bcl-2, bax or the bax:bcl-2 ratios are not predictive indicators of clinical sensitivity in CLL, but an increase in mdm-2 levels after drug treatment is indicative of p53 function in these cells.

In vitro cytotoxicity of 2-chlorodeoxyadenosine and chlorambucil in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is most commonly treated with the alkylating agent chlorambucil (CLB), although the nucleoside analogs, fludarabine (Flu) and 2-chlorodeoxyadenosine (CdA), are also effective in this disease. In this study, we investigated the in vitro cytotoxicity of CdA and CLB in CLL cells from 12 patients in vitro. Treatment with CLB for 6 h, followed by CdA for 18 h, resulted in 2.3- to 7.5-fold synergistic cytotoxicity in leukemic cells from 10 patients and an additive effect in cells from two patients. In general, synergy was greatest in patients who were sensitive to CLB or CdA, and could be enhanced by increasing the concentrations of CLB or CdA. Synergy was only observed if the cells were treated with CLB prior to CdA. Synergy could not be explained by an increase in the incorporation of CdA into DNA, or by the inhibition of repair of CLB-induced DNA crosslinks by CdA. In contrast to CLL cells, treatment of human marrow in vitro with CLB and CdA resulted in a low level of synergy for CFU-GM cells, and additive cell kill in erythroid progenitors. Thus, treatment with CdA and CLB can produce selective synergistic cell kill in CLL cells, and combination therapy may improve the therapeutic index of these agents in chemosensitive patients.

Comparison of antitumor activities of 2-chlorodeoxyadenosine and 9-beta-arabinosyl-2-fluoroadenine in chronic lymphocytic leukemia and marrow cells in vitro.

The in vitro antitumor activities of the nucleoside analogs, 2-chlorodeoxyadenosine (CdA) and 9-beta-arabinosyl-2-fluoroadenine monophosphate (Flu), and the alkylating agent, chlorambucil (CLB), were compared in leukemic cells from 28 patients with chronic lymphocytic leukemia (CLL). On a molar basis, the median sensitivities of the cells to these agents were CLB > CdA > Flu. CLL cells from 90% of the patients had similar relative orders of sensitivities to CdA and Flu, while cells from 10% of the patients showed differential sensitivities to these agents. There was no relationship between the sensitivities of the cells to the nucleoside analogs and sensitivity to CLB. CdA and CLB produced similar toxicities to human marrow progenitor cells in vitro, while Flu was less toxic to these cells. An 18 h exposure to CdA produced significantly greater cell kill of both CLL and marrow progenitor cells than an equivalent 2 h treatment; however, the difference in cytotoxicity was greater for the tumor cells resulting in a higher therapeutic index with the 18 h treatment. The intracellular accumulation of drug varied 5-fold for CdA, with the major metabolite being CdAMP, and 15-fold for Flu, with the major metabolite being F-ara-ATP. However, the accumulation of CdA, Flu or their metabolites did not predict for drug sensitivity. These studies suggest that CdA and Flu cross-resistance cannot be assumed in all CLL patients. The therapeutic effectiveness of CdA may be enhanced by use of a prolonged, low-dose drug regimen.

Combination therapy with nucleoside analogs and alkylating agents.

The nucleoside analog, 2'-deoxycoformycin (dCF), and the alkylating agents, chlorambucil (CLB) and cyclophosphamide, are effective agents in the treatment of chronic B cell leukemias and lymphomas. The cyclophosphamide analog, 4-hydroperoxycyclophosphamide (4-HC), generates the same active metabolite as cyclophosphamide in cells and has been used extensively for bone marrow purging in vitro. We have observed that deoxyadenosine (dAdo) plus dCF (dAdo/dCF) inhibit the repair of x-irradiation-induced and bleomycin-induced DNA damage in vitro, and that this results in either synergistic or additive cytotoxicity, respectively. In the present study we examined whether dAdo/dCF, can enhance the antitumor activity of CLB and 4-HC in chronic lymphocytic leukemia (CLL) cells in vitro. CLL cells were treated with CLB for 6 hr and then with dAdo/dCF for 18 hr and cytotoxicity was measured by the MTT assay. Synergy was observed between CLB and dAdo/dCF in CLL cells from 2 patients, with synergy increasing as the CLB dose was raised. In contrast, similar treatment of human bone marrow cells resulted in little or no synergistic cell kill. Treatment of CLL cells from 2 patients with 4-HC for 30 min followed by dAdo/dCF for 18 hr resulted in little synergistic cytotoxicity, although this drug combination did produce an additive cell kill. Thus, combination therapy with nucleoside analogs and alkylating agents may be useful for improving treatment of CLL.

Prevention of insulin-dependent diabetes mellitus by 2'-deoxycoformycin in the BB Wistar rat.

The effect of the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin (dCF) on the development of insulin-dependent diabetes mellitus (IDDM) was assessed in the BB Wistar rat. Sixty-one male rats were treated from days 30 to 120 with 0, 0.5, 1.0 or 1.5 mg dCF/kg/week. The incidence of IDDM was 78% in the controls and was significantly (P < 0.01) decreased in rats receiving 1.5 mg dCF/kg/week (32%), but not in rats receiving lower doses of the drug. However, for those rats that became diabetic the mean time to the development of IDDM was unchanged in animals receiving dCF compared with control. dCF treatment did not produce significant weight loss in the animals or gross changes in the thymus, spleen or kidneys. Although the protective effect of dCF against IDDM was likely produced by immunosuppression, the different dCF dosages had similar effects on ADA suppression in spleen or thymus and on dATP accumulation in these organs.

Role of transforming growth factor-beta in chronic lymphocytic leukemia.

TGF-beta is an important immunoregulator as it suppresses proliferation and function of B- and T-lymphocytes. In the present study we have examined the cellular localization and secretion of TGF-beta in B-cells from normal donors and patients with CLL and have assessed the influence of TGF-beta 1 on DNA synthesis in these cells. Using anti-LC(1-30)--a polyclonal anti-TGF-beta 1 antibody--TGF-beta was localized to discrete sites within the cytoplasm of both normal and malignant lymphocytes. These areas co-localized with areas detected by an antigranule antibody (D545), suggesting that TGF-beta may be stored within cytoplasmic secretory vesicles. Both normal B- and CLL cells contained low or undetectable levels of TGF-beta mRNA and secreted low and equivalent amounts of TGF-beta. Compared to untreated cells, DNA synthesis was reduced by TGF-beta 1 to a mean +/- S. E. of 0.84 +/- 0.07 in CLL cells and this was significantly less (p < 0.001) than that observed in normal B-cells (mean +/- S. E. of control, 0.12 +/- 0.02). In 3 of the 18 patients, TGF-beta 1 stimulated DNA synthesis. The reduced inhibition of leukemic cell DNA synthesis by TGF-beta 1 in CLL may provide these cells with a growth or survival advantage over normal lymphocytes and contribute to their selective accumulation.

Induction of apoptosis in CD4+ prolymphocytic leukemia by deoxyadenosine and 2'-deoxycoformycin.

The leukemic cells of a patient with CD4+ prolymphocytic leukemia were treated in vitro with 5 microM deoxyadenosine and 60 microM 2'-deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA). Following treatment, the leukemic cell dATP level increased to 378 pmol/10(6) cells on day 3, after which the level plateaued. Apoptosis was apparent following 4 h of incubation, and by day 8 34% of the chromatin was fragmented. Apoptosis also occurred in control cells, but to a lesser extent than in drug-treated cells. When the patient was treated with dCF, 4 mg/M2 i.v. the leukemic cell ADA activity was inhibited 24 h following treatment, and the lymphocyte dATP content increased to 303 pmol/10(6) cells by day 6. The lymphocyte count fell 60% in 1 week, but during this time there was no evidence of apoptosis in these cells. Thus, if dCF induces apoptosis in vivo, the effete cells may be rapidly cleared from the circulation and thus elude detection.

Factors influencing the inhibition of repair of irradiation-induced DNA damage by 2'-deoxycoformycin and deoxyadenosine.

Permeabilized L5178Y cells were used to investigate the mechanism underlying inhibition of the repair of irradiation-induced DNA strand breaks by 2'-deoxycoformycin combined with deoxyadenosine. Permeabilized cells repaired DNA strand breaks as effectively as did intact cells, and at deoxyadenosine concentrations that produced similar levels of deoxyadenosine triphosphate (dATP), repair of DNA strand breaks was inhibited by 2'-deoxycoformycin plus deoxyadenosine to a comparable extent in both types of cells. Accompanying the increase in intracellular dATP produced by 2'-deoxycoformycin combined with deoxyadenosine was a fall in levels of deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP). The addition of dTTP, dGTP, and dCTP reversed the inhibition of DNA repair by 2'-deoxycoformycin plus deoxyadenosine, although the level of dATP was not affected. Reducing the phosphorylation of deoxy-adenosine to dATP by the addition of adenosine prevented the decrease in levels of dTTP, dGTP, and dCTP and the inhibition of DNA repair by 2'-deoxycoformycin and deoxyadenosine. In contrast, increasing the intracellular levels of dATP by the addition of 2'-deoxycoformycin together with dATP, deoxyadenosine diphosphate (dADP), or deoxyadenosine monophosphate (dAMP) had no effect on the levels of the other deoxynucleotide triphosphates and did not inhibit DNA repair. Moreover, DNA repair was not inhibited by the breakdown products of deoxyadenosine, adenine, or deoxyribose. These results suggest that inhibition of the repair of irradiation-induced DNA strand breaks by 2'-deoxycoformycin combined with deoxyadenosine requires the phosphorylation of deoxyadenosine and involves alterations in the levels of deoxynucleotide triphosphates.

Glutathione S-transferase activity, sulfhydryl group and glutathione levels, and DNA cross-linking activity with chlorambucil in chronic lymphocytic leukemia.

Glutathione (GSH) levels and glutathione S-transferase (GST) activities were measured in the leukemia cells of 12 patients with chronic lymphocytic leukemia. Both were correlated with prior clinical exposure to alkylating agents and with DNA cross-link formation by chlorambucil in these cells in vitro. No correlation was observed between prior exposure to alkylating agents and GSH level or GST activity. An inverse correlation was observed between GST activity and cross-linking by chlorambucil, which was enhanced if both GST activity and GSH level were related to cross-linking. These findings suggest that the combination of GST and GSH protects the DNA of leukemia cells from chlorambucil, but the role of this combination in clinical resistance remains to be determined.